J. Agric. Food Chem. 2010, 58, 2703–2709
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DOI:10.1021/jf902543r
Synthesis and Antiviral Activities of Phenanthroindolizidine Alkaloids and Their Derivatives† KAILIANG WANG, BO SU, ZIWEN WANG, MENG WU, ZHENG LI, YANNA HU, ZHIJIN FAN, NA MI, AND QINGMIN WANG* State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, People’s Republic of China
Racemic phenanthroindolizidine alkaloids tylophorine, antofine, and deoxytylophorinine, and optically pure alkaloids S-(þ)-tylophorine and R-(-)-tylophorine were synthesized and evaluated for their antiviral activities against tobacco mosaic virus (TMV). Further salinization modifications based on tylophorine increased stability and water solubility and improved the antiviral activity in application. The bioassay results showed that most of these synthesized compounds showed higher antiviral activity against TMV in vitro and in vivo than commercial Ningnanmycin. Especially, tylophorine salt derivatives 10, 11, 13, 17, and 22 emerged as potential inhibitors of plant virus. These findings demonstrate that these phenanthroindolizidine alkaloids and their salt derivatives represent a new template for antiviral studies and could be considered for novel therapy against plant virus infection. KEYWORDS: Phenanthroindolizidine alkaloid; racemic alkaloids; optically pure alkaloids; salt derivatives; tobacco mosaic virus; inhibitors
INTRODUCTION
Plant viruses are unique in the deceptive simplicity of their structure. However, this simplicity leads to a greater dependence on the host; a highly intricate relationship exists between the two, which complicates the strategic designs to control plant viruses and the losses caused by them (1). The plant disease caused by tobacco mosaic virus (TMV) is found worldwide. TMV is known to infect members of 9 plant families, and at least 125 individual species, including tobacco, tomato, pepper, cucumbers, and a number of ornamental flowers. The amount of loss can vary from 5 to 90% depending on the strain of TMV, the total time of infection by TMV, the temperature during disease development, and the presence of other diseases. It is found that in certain fields 90-100% of the plants show mosic or leaf necrosis by harvesting time. Therefore, this plant virus has the name “plant cancer” and is difficult to control. Ningnanmycin, a commercial antiviral agent, isolated from Strepcomces noursei var. xichangensisn by the Chengdu Institute of Biology, Chinese Academy of Sciences, is a kind of microbial pesticide. It is more effective in the treatment of plants against TMV than other existing commercial agents. However, the use of this agent for field trial is largely limited by its photosensitivity and water stickiness (2). Therefore, further research needs to be conducted in this area for the development of a highly efficient, novel, environmentally benign antiviral inhibitor. Natural phenanthroindolizidine alkaloid tylophorine (Figure 1) and its analogues (e.g., antofine and deoxytylophorinine) have been
isolated primarily from the genera Cynanchum, Pergularia, and Tylophora in the Asclepiadaceae family (3). These compounds, commonly called tylophora alkaloids, have been targets of synthesis and modification for their significant cytotoxic activities (4). Evaluation of tylophora alkaloids in the National Cancer Institute’s antitumor screen showed a uniform and potent growth inhibitory effect (GI50 = 10-8 M) against all 60 cell lines, with notable selectivity toward several refractory cell lines, including melanoma and lung tumor cell lines (5). To date, most of the studies have been focused on anticancer activity in medicinal formulation. However, relatively little is known about the antiviral activity of tylophora alkaloids in pesticide formulation. In our preliminary work, (-)-antofine was isolated from the aerial parts of Cynanchum komarovii and was first found to have good antiviral activity against TMV in vitro (6). However, the content of natural antofine is especially low. Antofine also has the drawbacks of being easily decomposed in light and having poor water solubility. All of these limited its application in plant protection. To extend our research work to tylophora alkaloids as a new class of antiviral agents against TMV, we designed and synthesized three representative racemic alkaloids (tylophorine, antofine, and deoxytylophorinine), two optically pure alkaloids (S-(þ)-tylophorine and R-(-)-tylophorine), and a series of tylophorine salt derivates for antiviral activity evaluation. MATERIALS AND METHODS
Part of the ECUST-Qian Pesticide Cluster. *Author to whom correspondence should be addressed (telephone þ86-022-23499842; fax þ86-022-23499842; e-mail
[email protected],
[email protected]).
Synthetic Procedures. Reagents were purchased from commercial sources and were used as received. All anhydrous solvents were dried and purified by standard techniques just befor use. Reaction progress was monitored by thin-layer chromatography on silica gel GF-254 with detection by UV. Melting points were determined on an X-4 binocular
© 2009 American Chemical Society
Published on Web 12/10/2009
†
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Figure 1. Chemical structures of tylophora alkaloids. microscope melting point apparatus (Beijing Tech Instruments Co., Beijing, China) and are uncorrected. 1H NMR spectra were obtained at 300 MHz using a Bruker AC-P 300 and a Varain Mercury Plus 400 MHz spectrometer. Chemical shift values (δ) are given in parts per million and were downfield from internal tetramethylsilane. High-resolution mass spectra (HRMS) were recorded on FT-ICR MS (Ionspec, 7.0T).
solid: mp 236-238 °C; 1H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.77 (s, 1H), 7.62 (s, 1H), 7.41 (s, 1H), 7.17 (s, 1H), 5.50 (d, 2JHH = 14.4 Hz, 1H), 4.40 (d, 2JHH = 14.4 Hz, 1H), 4.12 (s, 6H), 4.03 (s, 6H), 3.84-3.87 (m, 1H), 3.58 (s, 3H), 2.53-2.66 (m, 1H), 2.33-2.46 (m, 1H), 1.92-2.19 (m, 2H). HRMS (ESI) m/z calcd for C25H27NO7Na (M þ Na)þ, 476.1680; found, 476.1680.
Synthesis of 2,3-Bis(3,4-dimethoxyphenyl)acrylic Acid (3).
Synthesis of (S)-(þ)-N-[(2,3,6,7-Tetramethoxy-9-phenanthryl)methyl]pyroglutamic Acid (7). Compound 6 (1.81 g, 4.0 mmol) was
3,4-Dimethoxybenzaldehyde (1) (30.0 g, 0.18 mol), 3,4-dimethoxyphenylacetic acid (2) (32.7 g, 0.17 mol), acetic anhydride (70 mL), and triethylamine (35 mL) were heated to reflux for 20 h with the exclusion of moisture. The solution was allowed to cool to room temperature, water (150 mL) was added, and the mixture was stirred for 1 h. The mixture was then poured into aqueous potassium carbonate (120.0 g in 300 mL water) and refluxed until nearly all of the gummy material had dissolved. The solution so obtained was cooled, extracted with ether (3 50 mL), and carefully acidified with concentrated hydrochloric acid (pH ≈1) to produce a white precipitate. The solid that separated was collected and washed from methanol (150 mL) to give 3 (47.0 g, 82.0%) as a mixture of E/Z isomers.
Synthesis of 2,3,6,7-Tetramethoxyphenanthrene-9-carboxylic Acid (4). To a stirred solution of the mixture of E-isomer 3 and Z-isomer 3 (24.1 g, 0.07 mol) in CH2Cl2 (500 mL) was added anhydrous FeCl3 (1.71 g, 0.01 mol) under nitrogen at 0-5 °C, and a solution of m-CPBA (12.1 g, 0.07 mol) in CH2Cl2 (100 mL) was added to the mixture, warmed to room temperature for 6 h, and quenched with methanol (100 mL). The solvents were concentrated in vacuo, and the residue was washed with methanol (200 mL), filtered, and washed with methanol (3 30 mL) again to give acid 4 (21.6 g, 90.0%) as a light yellow solid: mp 285-287 °C (lit. (7) 285-287 °C); 1H NMR (300 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.43(s, 1H), 8.03 (s, 1H), 7.99 (s, 1H), 7.54 (s, 1H), 4.08 (s, 3H), 4.07 (s, 3H), 3.94 (s, 3H), (s, 3H).
Synthesis of 2,3,6,7-Tetramethoxy-9-(hydroxymethyl)phenanthrene (5). To a mixture of LiAlH4 (1.14 g, 0.03 mol) in 150 mL of
THF at 0 °C was added 4 (6.84 g, 0.02 mol) in portions. The solution was refluxed for an additional 2 h, then brought back to 0 °C, at which point 10 mL of EtOAc was added dropwise, followed by 10 mL of 2 N HCl. The solution was stirred and filtered, and the solvent was removed by rotary evaporation to give 6.56 g (96.5%) of 5 as a white solid: mp 181-182 °C (lit. (8) 185 °C); 1H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.75 (s, 1H), 7.56 (s, 1H), 7.54 (s, 1H), 7.18 (s, 1H), 5.11 (s, 2H), 4.13 (s, 3H), 4.12 (s, 3H), 4.06 (s, 3H), 4.02 (s, 3H).
Synthesis of (S)-(þ)-N-[(2,3,6,7-Tetramethoxy-9-phenanthryl)methyl]pyroglutamic Acid Methyl Ester (6). Compound 5 (3.28 g,
10 mmol) was dissolved in 200 mL of CHCl3 and cooled to 0 °C. A solution of PBr3 (1.42 mL, 15 mmol) in 40 mL of CHCl3 was added dropwise under nitrogen. The solution was then stirred at room temperature for 4 h and poured over ice, and the two layers were separated. The organic phase was dried over Na2SO4, filtered, and concentrated in vacuo to afford a white solid. The solid was then redissolved in 280 mL of DMF. L-Glutamic acid dimethyl ester hydrochloride (BMPAC, 3.0 g, 14.2 mmol) was added and allowed to stir for 20 min. K2CO3 (2.0 g, 14.4 mmol) was added, and the mixture was allowed to stir at room temperature overnight. The solution was then rotary evaporated, and the product was partitioned between CHCl3 and H2O. The organic layer was dried over Na2SO4, filtered, and concentrated to obtain a crude product. The crude product was dissolved in 50 mL of MeOH and 20 mL of AcOH and stirred for 3 h at 45 °C. The solution was then evaporated, and the crude product was purified by flash column chromatography to give 2.5 g (55.0%) of 6 as a white
stirred in a solution of 50 mL of dioxane, 40 mL of MeOH, and 30 mL of 2 N KOH for 3 h. The solvents were concentrated, and 10 mL of water was added. The solution was cooled to 0 °C and acidified with concentrated hydrochloric acid (pH ≈1) to produce a white precipitate. The solid that separated was collected to give 7 (1.73 g, 98.5%) as a white solid: mp > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.01 (s, 1H), 7.97 (s, 1H), 7.47 (s, 1H), 7.44 (s, 1H), 7.36 (s, 1H), 5.40 (d, 2JHH = 14.8 Hz, 1H), 4.18 (d, 2JHH = 14.4 Hz, 1H), 4.01 (s, 6H), 3.89 (s, 3H), 3.85 (s, 3H), 3.65-3.70 (m, 1H), 2.27-2.45 (m, 2H), 2.06-2.20 (m, 1H), 1.81-1.94 (m, 1H).
Synthesis of (S)-(þ)-2,3,6,7-Tetramethoxyphenanthro[9,10-b]11,14-indolizidinedione (8). To a solution of 7 (1.1 g, 2.5 mmol) in 80 mL of CH2Cl2 was added freshly distilled oxalyl chloride (0.25 mL, 2.8 mmol) and 2 drops of DMF. The mixture was stirred for 2 h at room temperature and then warmed to reflux. SnCl4 (1.25 mL, 5.2 mmol) in 20 mL of CH2Cl2 was added, and the mixture was refluxed for an additional 6 h. The solution was cooled to room temperature, and 15 mL of cold 2 N HCl was added slowly. The phases were separated, and the organic phase was dried over Na2SO4 and filtered. The solvent was removed by rotary evaporation, and the crude product was purified by flash column chromatography to give 0.99 g (94.0%) of 8 as a yellow solid: mp 225-227 °C; (lit. (9) 228-230 °C); 1H NMR (400 MHz, CDCl3) δ 9.10 (s, 1H), 7.78 (s, 1H), 7.76 (s, 1H), 7.28 (s, 1H), 5.72 (d, 2JHH = 18.0 Hz, 1H), 4.69 (d, 2JHH = 17.6 Hz, 1H), 4.38-4.49 (m, 1H), 2.92 (d, 2JHH = 29.2 Hz, 1H), 2.48-2.71 (m, 3H).
Synthesis of (S)-(þ)-2,3,6,7-Tetramethoxyphenanthro[9,10-b]11-indolizidinone (9). To a solution of 8 (0.84 g, 2.0 mmol) in ethanol (100 mL) was added sodium borohydride(0.15 g, 4.0 mmol) in three portions at 0 °C. The mixture was warmed to room temperature in a period of 6 h. Ice-water (10 mL) was added, followed by a saturated aqueous solution of ammounium chloride (15 mL). The mixture was extracted with dichloromethane. The extracts were dried with MgSO4, filtered, and concentrated to obtain a white solid, which was used without further purification. To a solution of the solid in CH2Cl2 (20 mL) was added trifluoroacetic acid (10 mL) and triethylsilane (2 mL). The mixture was refluxed for 0.5 h, diluted with 15 mL of distilled water, and extracted with CH2Cl2 (50 mL). The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. Purification of the residue by flash column chromatography on silica gel gave 9 (0.76 g, 94%) as a light yellow solid: mp 236-238 °C; (lit. (9) mp 238-240 °C); 1H NMR (300 MHz, CDCl3) δ 7.83 (s, 1H), 7.82 (s, 1H), 7.27 (s, 1H), 7.15 (s, 1H), 5.31 (d, 2JHH = 17.4 Hz, 1H), 4.55 (d, 2JHH = 17.1 Hz, 1H), 4.12 (s, 6H), 4.06 (s, 3H), 4.04 (s, 3H), 3.87-4.00 (m, 1H), 3.43-3.50 (m, 1H), 2.77-2.91 (m, 1H), 2.48-2.64 (m, 3H), 1.96-2.09 (m, 1H). Synthesis of (S)-(þ)-Tylophorine. To a mixture of LiAlH4 (0.08 g, 2.0 mmol) in 35 mL of THF at 0 °C was added dropwise a solution of 9 (0.41 g, 1.0 mmol) in 15 mL of THF. The mixture was refluxed for 6 h and then brought back to 0 °C, at which point EtOAc (10 mL) was added dropwise, followed by H2O (5 mL). The solution was filtered and dried over MgSO4, and the solvent was removed by rotary evaporation to give 0.36 g (92.0%) of (S)-(þ)-tylophorine with 92% enantiomeric excess (ee)
Article value: mp 282 °C dec; (lit. (9) mp 282-284 °C dec); [R]20D þ100° (CHCl3); 1 H NMR (400 MHz, CDCl3) δ 7.84 (s, 1H), 7.83 (s, 1H), 7.32 (s, 1H), 7.17 (s, 1H), 4.64 (d, 2JHH = 14.4 Hz, 1H), 4.12 (s, 6H), 4.06 (s, 6H), 3.68 (d, 2 JHH = 14.4 Hz, 1H), 3.47-3.51 (m, 1H), 3.33-3.40 (m, 1H), 2.87-2.95 (m, 1H), 2.42-2.54 (m, 2H), 2.20-2.30 (m, 1H), 1.73-2.10 (m, 3H). Synthesis of (R)-(-)-Tylophorine. Following the same procedure as for (S)-(þ)-tylophorine gave optically pure alkaloid (R)-(-)-tylophorine with 96% ee value, [R]20D -93° (CHCl3). The optical purity of the synthesized alkaloids was determined via HPLC using an Agilent 1100 instrument with an AD-H column and gave 92 and 96% ee values, respectively. Detection was conducted at 254 nm. n-Hexane/isopropanol/triethylamine (75:25:0.025) was used as mobile phase at a flow rate of 1.0 mL/min.
General Procedure for the Preparation of Tylophorine Salt Derivatives 10-25. To a solution of 1.0 mmol of inorganic acid or organic acid in 50 mL of CHCl3 and 50 mL of CH3OH was added 1.0 mmol of racemic tylophorine in 50 mL of CHCl3 slowly under nitrogen at 40-50 °C. The mixture was refluxed for 2 h and allowed to stand for 2 h; the solvents were removed partially by rotary evaporation and filtered to obtain the corresponding salt derivatives 10-25. Data for 10: mp 258 °C dec; 1H NMR (400 MHz, DMSO-d6) δ 8.04 (s, 1H), 8.03 (s, 1H), 7.33 (s, 1H), 7.17 (s, 1H), 5.00-5.15 (m, 1H), 4.33-4.50 (m, 1H), 4.03 (s, 6H), 3.94 (s, 6H), 3.40-3.83 (m, 3H), 3.15-3.30 (m, 2H), 2.30-2.45 (m, 1H), 1.86-2.18 (m, 3H). Data for 11: mp 260 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.07 (s, 1H), 8.06 (s, 1H), 7.37 (s, 1H), 7.21 (s, 1H), 5.19-5.33 (m, 1H), 4.54-4.72 (m, 1H), 4.05 (s, 6H), 3.97(s, 6H), 3.64-3.91 (m, 3H), 3.15-3.48 (m, 2H), 2.53-2.64 (m, 1H), 1.86-2.31 (m, 3H). Data for 12: mp 240 °C dec; 1H NMR (400 MHz, DMSO-d6) δ 8.06 (s, 1H), 8.05 (s, 1H), 7.36 (s, 1H), 7.19 (s, 1H), 5.22-5.27 (m, 1H), 4.59-4.66 (m, 1H), 4.04 (s, 6H), 3.96 (s, 6H), 3.62-3.91 (m, 4H), 3.13-3.25 (m, 1H), 2.02-2.32 (m, 3H), 1.85-1.97 (m, 1H). Data for 13: mp 246 °C dec; 1H NMR (300 MHz, CDCl3) δ 8.02 (s, 1H), 8.00 (s, 1H), 7.82 (s, 2H), 7.45-7.51 (m, 1H), 7.30-7.40 (m, 3H), 7.14 (m, 1H), 4.86-4.91 (m, 1H), 4.12 (s, 3H), 4.11 (s, 3H), 4.06 (s, 3H), 4.04 (s, 3H), 3.97-4.00 (m, 1H), 3.65-3.76 (m, 1H), 3.38-3.48 (m, 1H), 3.173.27 (m, 1H), 3.00-3.12 (m, 1H), 2.77-2.90 (m, 1H), 2.31-2.42 (m, 1H), 1.90-2.20 (m, 3.H). Data for 14: mp 250 °C dec; 1H NMR (400 MHz, CDCl3) δ 7.80-7.85 (m, 2H), 7.21-7.26 (m, 1H), 7.02 (d, 1H), 5.37-5.57 (m, 1H), 4.78-4.93 (m, 1H), 4.44-4.60 (m, 1H), 4.04-4.15 (m, 12H), 3.55-3.65 (m, 1H), 3.32-3.44 (m, 1H), 3.08-3.20 (m, 2H), 2.66-2.69 (m, 1H), 2.48-2.56 (m, 1H), 2.37-2.46 (m, 1H), 2.13-2.30 (m, 2H), 1.89-1.91 (m, 1H), 1.641.80 (m, 4H), 1.46-1.56 (m, 1H), 1.02-1.11 (m, 1H), 0.88 (s, 3H), 0.62 (s, 3H). Data for 15: mp 225 °C dec; 1H NMR (400 MHz, DMSO-d6) δ 10.15 (br, 1H), 8.56 (s, 2H), 8.06 (s, 1H), 8.05 (s, 1H), 7.36 (s, 1H), 7.18 (s, 1H), 5.21-5.25 (m, 1H), 4.58-4.64 (m, 1H), 4.03 (s, 6H), 3.95 (s, 6H), 3.853.91 (s, 1H), 3.63-3.78 (m, 3H), 3.32-3.45 (m, 1H), 3.12-3.26 (m, 1H), 2.05-2.25 (m, 2H), 1.84-1.98 (m, 1H). Data for 16: mp 260 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.00 (s, 2H), 7.47 (d, 3JHH = 15.9 Hz, 1H), 7.19-7.32 (m, 4H), 7.05-7.09 (m, 1H), 6.79 (d, 3JHH = 8.1 Hz, 1H), 6.35 (d, 3JHH = 15.9 Hz, 1H), 4.56 (d, 2JHH = 15.6 Hz, 1H), 4.02 (s, 6H), 3.93 (s, 6H), 3.81 (s, 3H), 3.53 (d, 2JHH = 15.6 Hz, 1H), 3.32-3.37 (m, 2H), 2.71-2.89 (m, 1H), 2.30-2.40 (m, 2H), 2.10-2.24 (m, 1H), 1.58-1.95 (m, 3H). Data for 17: mp 260 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.03 (s, 2H), 7.35 (s, 1H), 7.20 (s, 1H), 4.75-4.81 (m, 1H), 4.20 (s, 4H), 4.07-4.14 (m, 1H), 4.03 (s, 6H), 3.94 (s, 6H), 3.48-3.54 (m, 2H), 2.67-3.00 (m, 3H), 2.21-2.35 (m, 1H), 1.67-2.03 (m, 3H). Data for 18: mp 241 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.06 (s, 2H), 7.66 (d, 3JHH = 7.5 Hz, 1H), 7.37 (s, 1H), 7.22 (s, 2H), 6.64-6.71 (m, 2H), 4.98-5.16 (m, 1H), 4.28-4.52 (m, 1H), 4.04 (s, 6H), 3.96 (s, 6H), 3.60-3.84 (m, 2H), 3.33-3.50 (m, 1H), 3.06-3.26 (m, 2H), 2.33-2.47 (m, 1H), 1.82-2.18 (m, 3H). Data for 19: mp 235 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 11.0 (br, 2H), 8.32 (s, 1H), 8.04 (s, 1H), 8.05 (s, 1H), 7.35 (s, 1H), 7.20 (s, 1H), 4.93 (d, 2JHH = 15.0 Hz, 1H), 4.12-4.29 (m, 1H), 4.04 (s, 6H), 3.95 (s, 6H), 3.50-3.70 (m, 2H), 2.90-3.27 (m, 3H), 2.58-2.67 (m, 4H), 2.27-2.44 (m, 1H), 1.73-2.13 (m, 3H).
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Data for 20: mp 240 °C dec; H NMR (300 MHz, DMSO-d6) δ 8.02 (s, 2H), 7.34 (s, 1H), 7.20 (s, 1H), 4.73 (d, 2JHH = 15.3 Hz, 1H), 4.07-4.15 (m, 1H), 4.03 (s, 6H), 3.94 (s, 6H), 3.42-3.52 (m, 2H), 2.53-2.95 (m, 5H), 2.37-2.44 (m, 1H), 2.21-2.34 (m, 1H), 1.88-2.05 (m, 2H), 1.66-1.82 (m, 1H). Data for 21: mp 268 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.01 (s, 2H), 7.32 (s, 1H), 7.19 (s, 1H), 4.59 (d, 2JHH = 15.0 Hz, 1H), 4.02 (s, 6H), 3.93 (s, 6H), 3.61 (d, 2JHH = 15.3 Hz, 1H), 3.34-3.40 (m, 2H), 2.76-2.85 (m, 1H), 2.41 (s, 6H), 2.12-2.28 (m, 1H), 1.59-1.97 (m, 3H). Data for 22: mp 230 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.28 (s, 1H), 8.01 (s, 1H), 7.80 (s, 1H), 7.77 (s, 1H), 7.33 (s, 1H), 7.19 (s, 1H), 6.83 (s, 1H), 6.80 (s, 1H), 4.56 (d, 2JHH = 15.3 Hz, 1H), 4.02 (s, 6H), 3.93 (s, 6H), 5.50 (d, 2JHH = 14.7 Hz, 1H), 3.32-3.41 (m, 2H), 2.70-2.85 (m, 1H), 2.33-2.45 (m, 2H), 2.10-2.25 (m, 1H), 1.57-2.00 (m, 3H). . Data for 23: mp 230 °C dec; 1H NMR (400 MHz, CDCl3) δ 7.81 (s, 2H), 7.28 (s, 1H), 7.11 (s, 1H), 4.71 (d, 2JHH = 14.8 Hz, 1H), 4.16-4.22 (m, 1H), 4.12 (s, 6H), 4.05 (s, 6H), 3.50-4.00 (m, 3H), 3.45-3.50 (m, 1H), 3.36-3.40 (m, 1H), 3.00-3.07 (m, 1H), 2.50-2.80 (m, 2H), 2.24-2.36 (m, 1H), 1.80-2.16 (m, 3H), 1.18-1.26 (m, 1H). Data for 24: mp 241 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 8.07 (s, 2H), 7.20-7.37 (m, 4H), 6.39-6.42 (m, 2H), 5.00-5.37 (m, 2H), 4.44-4.70 (m, 1H), 4.05 (s, 6H), 3.97 (s, 6H), 3.50-3.80 (m, 3H), 3.08-3.23 (m, 1H), 1.80-2.30 (m, 4H). Data for 25: mp 204-206 °C dec; 1H NMR (300 MHz, DMSO-d6) δ 10.00-10.30 (br, 1H), 8.03-8.08 (m, 3H), 7.65-7.68 (m, 1H), 7.37 (s, 1H), 7.20 (s, 1H), 6.84 (d, 3JHH = 8.4 Hz, 1H), 5.24 (d, 3JHH = 15.6 Hz, 1H), 4.55-4.72 (m, 1H), 4.05 (s, 6H), 3.97 (s, 6H), 3.60-3.90 (m, 3H), 3.10-3.50 (m, 3H), 1.83-2.32 (m, 3H). Antiviral Biological Assay. Purification of TMV. Using Gooding’s method (10), the upper leaves of Nicotiana tabacum L. inoculated with TMV were selected and ground in phosphate buffer and then filtered through double-layer pledget. The filtrate was centrifuged at 10000g, treated with PEG twice, and centrifuged again. The whole experiment was processed at 4 °C. Absorbance value was estimated at 260 nm by ultraviolet spectrophotometer. 1
0:1%;260nm virus concn ¼ ðA260 dilution ratioÞ=E 1cm
Antiviral Activity of Compounds against TMV in Vitro. In vitro activity of the synthesized compounds against TMV was performed by the conventional half-leaf method (11). Fresh leaf of the 5-6 growth stage of tobacco inoculated by the juice-leaf rubbing method (concentration of TMV is 5.88 10-2 μg/mL) was cut into halves along the main vein. The halves were immersed into the solution of different concentrations (see Table 1) of the compounds and double distilled water for 20 min, respectively, and then cultured at 25 °C for 72 h. Every concentration for each compound was replicated at least three times. Protective Effect of Compounds against TMV in Vivo. The compound solution was smeared on the left side and the solvent serving as control on the right side of growing N. tabacum L. leaves of the same ages. The leaves were then inoculated with the virus after 12 h. A brush was dipped in TMV of 6 10-3 mg/mL to inoculate the leaves, which were previously scattered with silicon carbide. The leaves were then washed with water and rubbed softly along the nervature once or twice. The local lesion numbers appearing 3-4 days after inoculation were counted (12). There are three replicates for each compound. Inactivation Effect of Compounds against TMV in Vivo. The virus was inhibited by mixing with the compound solution at the same volume for 30 min. The mixture was then inoculated on the left side of the leaves of N. tabacum L., whereas the right side of the leaves was inoculated with the mixture of solvent and the virus for control. The local lesion numbers were recorded 3-4 days after inoculation (12). There are three replicates for each compound. Curative Effect of Compounds against TMV in Vivo. Growing leaves of N. tabacum L. of the same ages were selected. TMV (concentration of 6.0 10-3 mg/mL) was dipped and inoculated on the whole leaves. Then the leaves were washed with water and dried. The compound solution was smeared on the left side, and the solvent was smeared on the right side for control. The local lesion numbers were then
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Wang et al.
Table 1. In Vitro Antiviral Activities against TMV compd 10
12
14
16
18
20
22
24
(()-tylophorine
(()-deoxytylophorinine
R-(-)-tylophorine
concn (μg/mL)
inhibition rate (%)
EC50 (μg/mL)
100 50 25 5 100 50 25 5 100 50 25 5 100 50 25 5 100 50 25 5 100 50 40 25 100 50 25 5 100 50 25 5 100 50 25 5 100 50 40 25 100 50 25 5
92.3 88.2 75.4 30.2 91.6 89.9 76.8 62.1 73.9 60.6 42.4 32.3 72.2 68.5 18.5 15.7 59.0 54.6 24.3 23.7 66.7 61.5 69.1 65.7 84.9 70.0 54.7 50.0 80.9 74.3 58.8 31.4 57.6 42.5 41.2 34.0 75.0 66.7 79.2 65.0 62.1 55.2 5.3 0
10.0
11
